The elevation of cytosolic Ca2+ is a ubiquitous signaling process in eukaryotic cells. Specificity of the physiological response initiated by the Ca2+ signal is in part encoded by the nature of the cytosolic Ca2+ increase. In plant cells, the cell pairs that regulate gaseous exchange in the leaf, specificity to various stimuli (light, temperature, abscisic acid, CO2) is thought to be encoded by spatial, temporal and amplitude (STA) variations in the Ca2+ signal. We plan to dissect these comples Ca2+ signals using single guard cell imaging utilizing the techniques available at NCMIR. Previous studies in the laboratory of Dr. J.I. Schroeder have shown that guard cells can be loaded with the Ca2+ -sensitive dyes Calcium green and Fura 2 by a non-invasive ester loading technique. This significant advance can now be utilized to study Ca2+ signals in these cells. Questions such as the source of Ca2+ (extracellular or intracellular), the STA nature of the signal and the possible oscillation of the signal will be addressed. The information obtained from the images obtained at NCMIR will be combined with concurrent experiments performed in the Schroeder laboratory that utilize electrophysiological techniques such as patch clamping and single electrode voltage clamp. The combination of these techniques will yield important information on the nature and regulation of the transport processes that underlie the generation of Ca2+ signals in guard cells. The work will use Arabidopsis thaliana guard cells. A. thaliana mutants defective in guard cell function have been isolated in the Schroeder laboratory. These mutants will be tested for variations in their CA2+ signaling phenotype, so specific mutations can be linked to defined roles in guard cell signaling. The mutant work will focus particularly on the hormone Absisic acid (ABA). ABA regulates guard cell turgor, and therefore, stomatal aperture by Ca2+-mediated regulation of ion channels. Many mutants are now available in the Schroeder laboratory which show aberrant ABA responses. Using these ABA mutants and an integration of the techniques available at NCMIR and in the Schroeder laboratory could yield important information on the mechanism of action of this important plant hormone. Work on this project is underway.